This project is aimed at understanding the molecular basis of intracellular iron metabolism. The cis and trans elements mediating the iron-dependent alterations in abundance of ferritin and the transferrin receptor have been identified and characterized in previous years in this laboratory. Iron-responsive elements (IREs) are RNA stem-loops found in the 5' end of ferritin mRNA and the 3' end of transferrin receptor mRNA. We have cloned, expressed, and characterized two essential iron-sensing proteins, Iron Regulatory Protein 1 (IRP1) and Iron Regulatory Protein 2 (IRP2), formerly referred to as iron-responsive element binding proteins (IRE-BPs). IRPs bind IRE's when iron levels are depleted, resulting in the inhibition of translation of ferritin mRNA and other IRE containing transcripts and prolongation of the half-life of the transferrin receptor mRNA. IRP1 is an iron-sulfur protein related to mitochondrial aconitase, a Krebs cycle enzyme, and it functions as a cytosolic aconitase in cells that are iron replete. Regulation of RNA binding activity of IRP1 involves a transition from a form of IRP1 in which a [4Fe-4S] cluster is bound, to a form that loses both iron and aconitase activity. The [4Fe-4S] containing protein does not bind IREs. Controlled degradation of the iron-sulfur cluster and mutagenesis reveals that the physiologically relevant form of the RNA binding protein in iron-depleted cells is apoprotein. The status of the cluster appears to be the key to determining whether IRP1 will bind RNA. Recently, we have identified mammalian enzymes of iron-sulfur cluster assembly that are homologous to the NifS and Nif U genes implicated in bacterial iron-sulfur cluster assembly, and we have shown that these gene products facilitate assembly of the iron-sulfur cluster of IRP1. IRP2 also binds IREs in iron-depleted cells, but unlike IRP1, IRP2 is rapidly degraded in cells that are iron-replete. An additional exon present in IRP2 determines whether IRP2 is subject to degradation and mutagenesis of cysteines within the 73 amino acid domain reveals that cysteines are required for degradation. We have recently shown that IRP2 is oxidatively modified in iron-replete cells, and that the modified intermediate is ubiquitinated prior to being degraded by the proteasome. Oxidative modification generates the recognition signal for ubiquitination in the case of IRP2, and indirect evidence suggests that numerous other proteins will be degraded by a pathway in which oxidative modification is followed by ubiquitination and proteasomal degradation of the ubiquitinated substrate. To approach questions about the physiology of iron metabolism, loss of function mutations of IRP1 and IRP2 have been generated in mice through homologous recombination in embryonic cell lines. There is no obvious phenotype associated with loss of IRP1 function, and the phenotyp of IRP2 loss of function mutations is presently being characterized. Creation of mice with loss of function mutations of both IRP1 and IRP2 are underway. Characterization of the roles of the genes for hereditary hemochromatosis and murine microcytic anemia are ongoing in normal and knockout mice.